Technical Abstract:
Peanut is one of the principle legume crops. Fungi in the genus Aspergillus produce aflatoxins as secondary metabolites which are highly poisonous and carcinogenic. Leaf spots caused by Cercospora arachidicola and Cercosporicium personatum are among the worst foliar diseases of peanut. To identify important host resistant gene and to understand the resistant mechanism, we conducted research using EST cDNA libraries, microarray screening and real time PCR techniques. Thousands of ESTs from two libraries were sequenced, and 380 clones were selected as putative resistance genes against diseases and drought-stress from the two libraries and used to produce cDNA microarray chips. The first batch of 1345 ESTs has been submitted to GenBank (CD037499 to CD038843) and recently 278 SSRs (simple sequence repeats) in peanut (AY526357 to AY526456, AY731521 to AY731698) have been released to GenBank from sequencing 5000 clones of two SSR enriched genomic libraries. We are reporting here the progress on identification of genes associated with disease resistance and drought tolerance.
We used cDNA microarray, which partly came from the A.flavus and drought induced cDNA library for resistant genotype A13, to screen the two stresses induced genes. About 83 up-regulated spots (Log2 ratio>1) under the fungal and drought stresses, and 104 up-regulated spots in drought stress were detected by microarray analysis. Among the total up-regulated spots, forty-nine spots had positive results in both treatments. The top 20 genes expressed under the two stresses were chosen to validate the expression level of the screened genes, and studied their relativity with the tolerant traits using real-time PCR. Some known function genes which had ever been proved as drought or disease tolerant gene in other plants were validated to have differential expression in the peanut genotype A13 under the A.flavus inoculation and drought stress.
To identify transcripts of disease resistance to late leaf spot disease caused by Cercosporidium personatum in two peanut cultivars, C34-24 (resistant) and GT-YY20 (susceptible), a total of 112 spots representing 56 genes in several functional categories were detected to be up-regulated genes (Log2 ratio>1). Seventeen of the top 20 genes, each matching known function genes in GenBank, were selected for validation of their expression levels using real-time PCR. These two peanut cultivars, one resistant and one susceptible to late leaf spot disease, were also used to study the functional analysis of these genes and possibly link the genes to these traits of disease resistance. Microarray technology and real-time PCR were used for comparison of gene expression. Some genes identified by microarray analysis were validated by real-time PCR, and had significantly higher expression in the resistant cultivar challenged by C. personatum than the expression levels in the susceptible cultivar. Further investigations are needed to characterize each of these genes for their possible functions in disease resistance. Molecular markers could then be developed to more rapidly select for these genes in peanut breeding programs.